In vitro synthesis of adenovirus type 5 T antigens. I. Translation of early region 1-specific rna from lytically infected cells

Proteins encoded in regions EIA and EIB of human adenoviruses cause transformation of rodent cells. One protein from EIA also stimulates transcription of other early regions at early times in a productive infection. In the past, direct analysis of these proteins synthesized in vivo has been difficult because of the low levels produced in both transformed cells and productively infected cells. We present a simple method which leads to expression of EIA and EIB mRNAs and proteins at 30-fold greater levels than those observed during the early phase of a standard productive infection. Under these conditions, these proteins are among the most prominent translation products of infected cells. This allowed direct visualization of EIA and EIB proteins on two-dimensional gels of pulse-labeled total cell protein. Experiments with EIA and EIB mutants confirm that the identified proteins are indeed encoded in these regions. Two EIA proteins are observed, one translated from each of the major early EIA mRNAs. Both of these EIA proteins are phosphorylated.

Section: Discussionmentioning

confidence: 99%

Enhanced expression of adenovirus transforming proteins

Gaynor

Tsukamoto

Montell

et al. 1982

“…Fig. 1 [9]). It can be seen that the predominant product was a 19K protein and that, in addition, an appreciable amount of the 65K protein was made.…”

mentioning

confidence: 95%

“…A comparison with the proteins immunoprecipitated with anti-T serum from this XhoI-C-transformed cell line shows that the in vitro-synthesized 65K protein has an electrophoretic mobility identical to that of the in vivo-synthesized product but that the relative amounts of 19K and 65K are reversed. Proteins with similar electrophoretic mobility, including a 65K protein, were obtained with early viral RNA from lytically infected cells (9). Proteins synthesized in vitro by a wheat germ cell-free extract, programmed with RNA isolated from Ad5-transformed human embryonic cells (293.C31 cells) (5).…”

mentioning

confidence: 99%

In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA

Jochemsen¹,

Hertoghs²,

Lupker³

et al. 1981

Self Cite

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transforned by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.5%). In addition, the adenovirus type 5-transformed human embryonic kidney line 293.C31 was investigated. The products were immunoprecipitated with serum from tumor-bearing hamsters and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The results show that all transformed cells investigated contain early region la-specific RNAs which can be translated into proteins with molecular weights of 34,000 (34K), 36K, 40K, and 42K. Transformed cells that also contain an intact early region lb synthesized RNA which can be translated into proteins with molecular weights of 19K and 65K. Minor proteins of 15K, 16K, 17.5K, 18K, 25K, and 29K were also observed, but these proteins could not be mapped unambiguously. Cells transformed by the 8% HindIII-G apparently lack RNA encoding the 65K protein, but they do contain RNA coding for the 19K protein.

“…The Elb gene codes for 55-and 21-kd polypeptides (15,24), whose functions during the viral replicative cycle remain unclear. The 55-kd protein has been shown to be a product of spliced 23S mRNA (15,38), whereas the 21-kd protein can be translated from either 23S or 13S mRNAs (5). Tryptic peptide analysis of both proteins revealed that they are unrelated (6), which Bos et al (5) confirmed by DNA sequence analysis.…”

mentioning

confidence: 98%

Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis

Babiss¹,

Ginsberg²

1984

174

To determine the role adenovirus 5 early region lb-encoded 21-and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl3l4 (A3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (O to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region lb functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dlll8, H5dlllO, H5inl27, and H5dll63) indicates that all of the viruses contain mutations which affect the 55kilodalton protein, whereas d1118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the^Thutants which is phenotypically identical to wild-type adenovirus 5). When compared with * Corresponding author. MATERIALS AND METHODS Cells and viruses. Monolayer cultures of human KB, 293, (21), HeLa, and KB cell line 18 (a KB cell line that constitutively expresses the bacterial XGPRT gene and the Ad2 Elb gene; 2) cells were grown in Dulbecco modified Eagle medium supplemented with 10% calf serum. Suspension cultures of human KB cells were grown in modified 202